Just Say “Nootropic”: The Effects of Nicotine on Memory ...

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Just Say “Nootropic”: The Effects of Nicotine onMemory and LearningElyse N. GoveiaConnecticut College, [email protected]

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Effects of Nicotine 1

Running head: THE EFFECTS OF NICOTINE ON MEMORY AND LEARNING

Just Say “Nootropic”: The Effects of Nicotine on Memory and Learning

in Adolescent and Adult Rats

A Senior Thesis presented by

Elyse N. Goveia

Connecticut College

New London, CT

May 2, 2008


Acknowledgements

First and foremost, I would like to thank my adviser, Dr. Ruth Grahn, for all of

her guidance and support throughout the year. Professor Grahn, I cannot even begin to

express how grateful I am to you for putting such a tremendous amount of time and effort

into this project, and for ensuring that I learned as much as possible along the way. You

helped make this one of the most rewarding experiences I have had during my time at

Connecticut College.

To my readers, Dr. Joseph Schroeder and Dr. Stuart Vyse, thank you so much for

your support and your valuable input—I could not have made it through the last leg of

this project without you.

I would also like to thank my family and friends for their endless encouragement

and support throughout the year.

And, last but not least, I would like to thank my rats, without whom this research

would not have been possible.


Abstract

This study investigated the effects of nicotine on memory and learning in adolescent and

adult male Fischer-344 rats. Rats were given 0.2 mg/kg/day of either nicotine or saline

chronically for 2 weeks and were tested in the Morris water maze as adolescents (Phase

1) and then again 4 months later as adults (Phase 2). There were 4 main groups:

nicotine/nicotine, nicotine/saline, saline/nicotine, and saline/saline. In Phase 2 rats were

tested for c-Fos and BrdU expression in the dentate gyrus of the hippocampus.

Behavioral data indicated that as adults, rats given nicotine were significantly improved

at the water maze task compared to rats given saline. Impairments of the S/N group

suggest performance in the maze is state dependent upon the nicotine. An increase in c-

Fos expression was seen in the saline rats, and no BrdU expression was seen in either

group. The behavioral results imply that low doses of nicotine improve learning and

memory in adult rats. This provides support for studies investigating nicotine as a

therapeutic agent for diseases affecting cognition, such as Alzheimer’s.


Table of Contents

Acknowledgements..…………………………………….……………….………..………2

Abstract…………..……………………………………………………………….…….…3

Introduction………..……………………………………….………………………...……5

Method…………………………………………...………………………………………29

Results……………………………………………………………………………………39

Discussion…………………………………………………………………………..……48

References…………………………………………………………………………..……67

List of Figures……………………………………………………………………………80


Just Say “Nootropic”: The Effects of Nicotine on Memory and Learning

in Adolescent and Adult Rats

For the past twenty years, nicotine and drugs that act as agonists to nicotinic

receptors have been under investigation due to their cognitive enhancing properties and

their potential for therapeutic use in certain neurodegenerative diseases, schizophrenia,

and attention deficit hyperactivity disorder (Dunbar et al., 2007; Levin & Rezvani, 2000;

Rezvani & Levin, 2001; White & Levin, 1999, , 2004). At the present time, the drugs

available to patients suffering from Alzheimer’s disease and other diseases affecting

cognition provide minimal relief of symptoms. Most of these drugs involve the indirect

stimulation of cholinergic receptors, however studies have shown that direct stimulation

of nicotinic receptors—via nicotine itself or nicotinic agonists—may have a more

pronounced therapeutic effect (Ono, Hasegawa, Yamada, & Naiki, 2002; Rezvani &

Levin, 2001). Thus, nicotine research has provided some promising results regarding

possible therapies for such diseases.

Nicotine: Neural Mechanisms

Nicotine, the main psychoactive ingredient found in tobacco, mimics the effects

of acetylcholine (ACh) at nicotinic ACh receptors (nAChRs). Like ACh, it modulates the

signaling pathways of target neurons by stimulating the release of several

neurotransmitters that are associated with memory, learning, and reward, including

acetylcholine, glutamate, dopamine, norepinephrine, and serotonin (Fagen, Mansvelder,

Keath, & McGehee, 2003; Levin, Limpuangthip, Rachakonda, & Peterson, 2006;

Mansvelder, van Aerde, Couey, & Brussaard, 2006; Rezvani & Levin, 2001; Wang et al.,

2008). It has been confirmed by multiple studies that nicotine has a pronounced effect on


areas of the brain linked with reward, specifically the mesolimbic system, through the

increased release of dopamine (Clarke, 1992; Di Chiara, Acquas, Tanda, & Cadoni,

1992). This is the essence of the controversy surrounding nicotine as a therapeutic drug:

its stimulation of receptors that release reward-based neurotransmitters could lead to

addiction, particularly if the dose is high and self-administration is possible (Merlo Pich,

Chiamulera, & Carboni, 1999; Wang et al., 2008).

Despite this controversy, addiction studies of nicotine provide some important

information that leaves the door open for nicotine’s use in diseases affecting cognition.

The most common way people become addicted to nicotine is via cigarette smoking

(Winger, Woods, & Hofmann, 2004). However, when nicotine is introduced into the

body in this way, a large amount of nicotine reaches the brain extremely rapidly on

inhalation. The immediate, semi-euphoric effect that results is what increases the

likelihood for nicotine addiction (Winger et al., 2004). Additionally, studies involving

self-administration of nicotine in rats have shown that when rats are allowed to self-

administer, they take in doses of nicotine on par with that of a cigarette smoker, which

contributes to research labeling it as addictive (Abrous et al., 2002). Evidence suggests

that when nicotine is administered to animals (or humans) at a slow rate and in a

controlled manner (not through cigarettes or tobacco), the possibility for addiction is

much lower (Samaha, Yau, Yang, & Robinson, 2005). Thus, as long as the dose of

nicotine is low and patients are on a well-defined schedule, it is less likely to produce an

addiction and has the potential to be a safe therapeutic alternative.

Though nicotine is the main rewarding component in cigarettes and the reason

smoking can be addictive, it is important to understand that the harmful effects of


smoking are largely due to the combination of an estimated 4000 constituents of cigarette

smoke—not the nicotine itself (Winger et al., 2004). When the smoke turns to vapor it

has high amounts of toxic carbon monoxide, which adds to the harmful effects of

cigarette smoking (Winger et al., 2004). Because of the toxic nature of the ingredients in

cigarettes and their reactions to being burned, it is no surprise that cigarette smoking can

lead to serious illnesses such as coronary heart disease, emphysema and various forms of

cancer (Winger et al., 2004). Nicotine alone, especially when administered intravenously

or by transdermal patches, does not cause these health problems (Winger et al., 2004).

The action of nicotine in the brain relies upon nAChRs. These receptors are

ligand-gated ion channels containing five subunits that form a circle around a central

pore, and allow the influx of positively charged ions. Subunits can be either alpha, beta,

gamma, or delta, but the subunits most connected to nicotinic involvement are alpha 4

beta 2 (α4β2) subunits or alpha 7 (α7) subunits (Hawi & Lowe, 2007; Winger et al.,

2004). When a nicotine molecule binds to a receptor, the receptor opens and allows

positively charged ions to enter. This leads to an excitatory postsynaptic potential

(EPSP), which increases the likelihood of neurotransmitter release from the responding

cell. It is this potential for neurotransmitter release that leads to nicotine’s cognitive

enhancing effects. The makeup of the subunits of the receptor as well as the location of

the receptor in the brain dictates the effect of the neurotransmitter release within the brain

(Winger et al., 2004).

Mouse studies of nicotine’s effect on gene expression and receptors have reported

that nicotine administration results in receptor upregulation in the nucleus accumbens

(one area from which nicotine is known to stimulate the release of dopamine), as well as


in the hippocampus, which is highly involved in learning and memory (Wang et al.,

2008). Receptor upregulation is an increase in available receptors in the brain that can

occur in response to a large amount of drug present in that brain area. In order to provide

a place for the extra drug molecules to bind, receptors multiply—this results in an

increased effect of the drug, and also leads to the potential for withdrawal symptoms if

the drug is suddenly ceased. When receptors are exposed to a large amount a drug,

however, receptors can become desensitized and receptor downregulation can occur as a

compensatory mechanism (Benwell, Balfour, & Birrell, 1995).

With regard to nicotine’s action upon certain receptors, much promising data has

come from studies concerning nicotinic cholinergic receptors in rats and mice. The α7

and α4β2 receptors, in particular, have been demonstrated to play a role in cognitive

functioning (Levin, McClernon, & Rezvani, 2006). A study using α7- nAChR knockout

mice determined that mice missing these receptors performed poorly on attentional tasks

of cognitive function when compared to normal controls (Young et al., 2007).

Experiments performed with rats in a passive avoidance test also demonstrated that both

chronic and acute nicotine administration had cognitive-enhancing effects as a result of

α7-nAChR stimulation in the hippocampus (Uzum, Diler, Bahcekapili, Tasyurekli, &

Ziylan, 2004). Wild-type mice with α7-nAChRs have also demonstrated more cell

proliferation as a result of nicotine administration when compared to mice lacking these

receptors (Koike et al., 2004).

The Hippocampus

Multiple studies have shown that nicotine has a positive effect on memory and

learning in humans and non-human animals, and one of the areas of the brain most


affected by nicotine administration is the hippocampus (Levin, Sledge, Baruah, & Addy,

2003; Wang et al., 2008). The hippocampus plays a key role in learning and memory,

particularly learning and memory involving spatial information (Izquierdo, 1995). Long

Term Potentiation (LTP) is the mechanism that allows for the hippocampus’ involvement

in memory storage and memory retrieval (Izquierdo, 1995). LTP is an increase in

synaptic effectiveness, which can result in changes in the synapses, an increase in

synaptic firing and an increase in neurotransmission in the hippocampus (Schumann et

al., 2004). Spatial learning tasks such as the Morris water maze can increase LTP in the

rat hippocampus, and it is possible that nicotine can also facilitate this process in

conjunction with these tasks.

One group has proposed that glutamate release in the hippocampus is enhanced by

nicotine. They examined the effect of nicotine and a glutamate agonist (dizocilpine) on

spatial learning in the radial arm maze. When the rats were given dizocilpine in

conjunction with nicotine, they displayed an impairment in working memory in the maze

(Levin et al., 2003). The dizocilpine was administered via a local infusion cannula in the

ventral hippocampus, so the impairment in working memory was localized specifically to

this brain area (Levin et al., 2003). This result provides support for the notion that the

hippocampus is an integral brain area in the formation of working memory, and that

glutamate neurotransmission as a result of nicotine administration is central to the

cognitive enhancing properties of nicotine.

Cognitive Enhancement in Humans

There have been numerous studies investigating nicotine’s effect on human

cognition. It has been shown that when regular smokers are deprived of cigarettes they


experience withdrawal symptoms that include cognitive deficits, such as decreased

accuracy on tasks of performance, and a decrease in psychomotor skills (Hatsukami,

Fletcher, Morgan, Keenan, & Amble, 1989). One study demonstrated that as little as 24

hours after cigarette cessation, reaction times and errors related to vigilance tasks were

significantly increased (Hatsukami et al., 1989). Though it is possible that the cognitive

decline could have been due in part to the other symptoms of withdrawal from cigarettes

(including headaches, nausea, etc.), it is likely that the decline was at least partially a

result of the cessation of nicotine. In 24 hours the participants in the study were not

reported to have any other noticeable withdrawal symptoms, which supports the notion

that the removal of nicotine was a contributing factor to the decrease in performance on

the cognitive tasks.

Due to the potential for cognitive decline following nicotine cessation in cigarette

smokers, it is beneficial to investigate the effect of nicotine on adult non-smokers without

any attentional or cognitive deficits—this research demonstrates nicotine’s effectiveness

in improving cognition in normal, healthy controls. Non-smokers are an ideal baseline in

these types of studies because there is no possibility of withdrawal, which could skew the

results. Additionally, regular smokers would have to change their smoking patterns for

the study, which would introduce even more potential for variation in the results

(Hatsukami et al., 1989; Levin & Rezvani, 2000; Rezvani & Levin, 2001). Nicotine has

been shown to improve the attention of non-smoking normal controls when administered

through subcutaneous injections and transdermal nicotine patches, suggesting that it may

also have a similar effect on patients with attentional disorders or other cognitive

impairments (Levin, Conners et al., 1998; Levin & Rezvani, 2000).


Alzheimer’s Disease

Because nicotine enhances the cognition of human non-smokers, it has been of

great interest to researchers whether it could have cognitive enhancing—and therefore

therapeutic—effects on people with various cognitive ailments, including Alzheimer’s

disease. Alzheimer’s disease (AD) is characterized by severe cognitive deficits,

including impairments of memory and executive (higher-order) function (Nixon, 2002).

Generally these deficits begin mildly, and become progressively worse as increased cell

death leads to severe cortical atrophy (Nixon, 2002). The most widely understood

neuropathology of post-mortem Alzheimer’s brains includes the presence of

neurofribrillary tangles and neuritic plaques (Buckner, Head, & Lustig, 2006; Nixon,

2002). The neuritic plaques are found largely in brain areas involved in learning and

memory, such as the neocortex, the hippocampus, and the amygdala. This provides

insight into Alzheimer’s negative effect on cognition as the disease progresses (Nixon,

2002).

In 2002, Ono and colleagues demonstrated that nicotine interferes with the

formation of in vitro amyloid plaques. This provides important information regarding the

neuroprotective effects of nicotine that extend beyond nicotinic receptor upregulation,

and supports the notion that nicotine can be used as a therapeutic agent in Alzheimer’s

disease. Studies have also shown that the neuropathology of postmortem Alzheimer’s

brains involves a significant decrease in nAChRs compared to that of normal ageing

(Court et al., 2001). Because nicotine administration results in an upregulation of

nAChRs, this effect could help delay some of the more severe cognitive deficits of the

disease by keeping the nAChR level in the brain stable and increasing neurotransmission.


Studies of nicotine’s effects in humans are generally carried out using injections,

nasal sprays, or transdermal nicotine patches. These methods of nicotine administration

cause nicotine to reach the brain much more slowly and are less likely to lead to a

nicotine addiction than smoking, in which nicotine reaches the brain extremely rapidly

(Samaha et al., 2005). The first study investigating the effects of intravenous nicotine

administration on Alzheimer’s patients used extremely small doses of nicotine, and found

that even when the dose was as small as 0.25 μg/kg/min, AD patients showed decreased

errors on a test of cognitive function (Newhouse et al., 1988). In studies using acute

nicotine doses of 0.4 to 0.8 mg, significant cognitive enhancement was demonstrated in

normal controls as well as Alzheimer’s patients on various tests of attention and cognitive

function (Jones, Sahakian, Levy, Warburton, & Gray, 1992; Sahakian, Jones, Levy, Gray,

& Warburton, 1989). More recently, however, the transdermal nicotine patch has been

used in order to provide a larger dose and to avoid the necessity of injections. In a pilot

study demonstrating the effects of the nicotine patch in AD patients, Wilson and

colleagues (1995) demonstrated that the nicotine group (still receiving a moderate dose—

0.9 mg/hr) showed improved learning. This was a result that had not been previously

reported (early studies only showed improved attention), and it paved the way for further

research in this area (Wilson et al., 1995).

In 1999, White and Levin investigated the effect of a 5 mg transdermal nicotine

patch when worn by patients with mild to moderate Alzheimer’s disease, and found that

the nicotine significantly improved patients’ performance on a task of attention, but there

were no significant differences relating to motor or memory function (White & Levin,

1999). Later, White and Levin (2004) performed a similar study using 5 mg transdermal


nicotine patches on patients with age-associated memory impairment (AAMI), which has

milder cognitive deficits than AD. Using the same scale as in the previous study (the

Conners’ Continuous Performance Test), they were able to demonstrate the same increase

in attention as a result of the nicotine administration in AAMI patients (White & Levin,

2004). Though in White and Levin’s studies no significant effects were seen in memory,

studies using nicotinic receptor agonists that report an improvement in declarative

memory, as well as the Wilson et al. (1995) study that showed improved learning,

provide evidence that nicotine can help alleviate the memory deficits found in AD and

other diseases affecting cognition (Potter et al., 1999).

Parkinson’s Disease

In addition to nicotine’s positive effect on patients suffering from diseases that

primarily affect cognition, nicotine has also been shown to be therapeutic in Parkinson’s

disease (PD), a neurodegenerative disease characterized by impairments in motor

coordination and function (Quik, 2004). The neuropathology of PD involves damage to

dopaminergic nigrostriatal neurons. This leads to the motor deficits of the disease, which

include tremor, rigidity, akinesia, bradykinesia, and gait disturbances (Mazziotta, Toga,

& Frackowiak, 2000; Quik, 2004). Though not as prevalent as the motor symptoms,

patients with Parkinson’s disease can also suffer from dementia, which is one of the

reasons nicotine has been investigated as a potential therapeutic agent (Quik, 2004). This

branch of research was first investigated after a series of studies emerged suggesting that

Parkinson’s disease is less prevalent in cigarette smokers, and that smoking can

potentially delay the onset of the disease (Fujita et al., 2006; Quik, 2004). It was thought

that the nicotine in cigarettes played a key role in this delay due to its stimulation of


dopaminergic nigrostriatal neurons, and its neuroprotective effects (Quik, 2004). In

addition to this, a neuroimaging study comparing nAChR expression in normal controls

vs. Parkinson’s patients concluded that patients with PD display a significant decrease in

nAChRs, and that nicotine could be beneficial to these patients as well as patients with

other neurodegenerative disease because it stimulates these receptors (Fujita et al., 2006).

A longitudinal study investigating the effects of high-dose nicotine administered

via transdermal patches on Parkinson’s patients confirmed the hypothesis that nicotine

can be beneficial to such patients (Villafane et al., 2007). Patients were given increasing

daily doses of nicotine (5-105 mg/day) for 14 weeks, keeping the highest tolerated dose

constant for at least 4 weeks before gradually decreasing the dose until the conclusion of

the experiment at week 29. Though most patients experienced symptoms of nausea and

insomnia, all patients demonstrated improved motor ability and required less of their

usually prescribed dopaminergic drugs by the end of the study (Villafane et al., 2007).

The ideal daily dose was determined to be between 45 and 90 mg/day, and because this

study was done over such a long duration, it provides convincing findings regarding

nicotine’s potential as a therapeutic agent for the motor symptoms of Parkinson’s disease

(Villafane et al., 2007).

Schizophrenia

Schizophrenia, a type of recurring psychosis that can have detrimental effects on

cognition, emotion, perceptions, and even motor function, is another illness that has long

been known to have a therapeutic tie to nicotine (Kraly, 2006; Postma et al., 2006).

Schizophrenics often show much higher rates of cigarette smoking when compared to the

general population, so studies involving nicotine and schizophrenia can be very useful in


order to better understand the disease (Postma et al., 2006; Smith, Singh, Infante,

Khandat, & Kloos, 2002). Schizophrenics have difficulty ignoring irrelevant sensory

input, and often show impairment in acoustic prepulse inhibition (PPI). PPI is

characterized by a decrease in the magnitude of response to a startling stimulus when the

stimulus is preceded by a lower intensity, non-startling stimulus (Postma et al., 2006). In

schizophrenic patients, the reaction to the startling stimulus is not lessened by the

prepulse, thus causing an overload of information that would not occur in normal patients

(Kumari, Soni, Mathew, & Sharma, 2000).

Nicotine has been shown to enhance PPI in both normal controls and

schizophrenic patients, and a functional magnetic resonance imaging (fMRI) study

showed that the increase in PPI as a result of the nicotine appeared largely in the

hippocampus (Postma et al., 2006). In addition to improved PPI, nicotine administered

via nasal spray has been demonstrated to improve cognition (through verbal memory

tasks and spatial organization tasks) in schizophrenics and has also led to improved

performance on oculomotor and attention tasks (Depatie et al., 2002; Smith et al., 2002).

These results suggest that the increase in smoking in schizophrenic patients is possibly a

form of self-medication, and provides evidence for a schizophrenia treatment involving

another method of nicotine administration that does not include the added risks associated

with cigarette smoking.

Attention Deficit/Hyperactivity Disorder

Because of the clear evidence that nicotine has a positive effect on attention,

studies have also been carried out to demonstrate nicotine’s effect on adults with

Attention Deficit/Hyperactivity Disorder (ADHD). ADHD is characterized by symptoms


of inattention and hyperactivity that are severe enough to interfere with daily life

(Sharkey & Fitzgerald, 2007). As would be expected, administration of nicotine to

ADHD patients leads to improved performance on tasks of attention, such as the

Conners’ Continuous Performance Test (CPT), and has also been reported to decrease

reports of depressed mood (Levin et al., 1996; Rezvani & Levin, 2001). Nicotine patches

administering 7 mg of nicotine to non-smoking ADHD patients showed an overall

nicotine-induced improvement on a modified version of the Clinical Global Impressions

(CGI) scale, as well as improving scores on the CPT (Levin et al., 1996).

From the numerous studies investigating the administration of nicotine to people

with diseases affecting different aspects of cognition, it can be inferred that nicotine can

be extremely beneficial to people suffering from a variety of illnesses. In illnesses like

Parkinson’s disease, schizophrenia and ADHD, adult patients have been shown to self

medicate with cigarettes, and evidence points to this being due to the nicotine they

receive (Levin, McClernon et al., 2006). Smoking, however, adds numerous risks due to

the toxins found in the smoke that cause lung damage and cancer, as well as the addictive

properties of nicotine when delivered in such high doses (Winger et al., 2004). Thus, if

nicotine can be administered in a more controlled and safe manner as a therapeutic agent,

patients would not have to be subjected to the risks associated with cigarette smoking.

Animal Models and Working Memory

Due to experimental limitations in human nicotine studies, much of the

preliminary and ongoing research regarding nicotine’s effects on cognition has come

from studies of non-human animals. Nicotine has been shown to have a significant effect

on cognition in non-human animals in various behavioral tasks that demonstrate working


memory. Working memory is an updated term for short-term memory, and it involves

the storage of information that is currently in use, or that is likely to be needed in a short

amount of time (Hegarty & Waller, 2005; Schumann et al., 2004). Working memory can

include verbal (in humans), object, and spatial tasks, and it is relatively easy to test this

type of memory function in lab animals using object recognition tasks, the Morris Water

Maze, or the Radial Arm Maze (Hegarty & Waller, 2005; Schumann et al., 2004). An

example of nicotine’s effect on working memory can be demonstrated by the

performance of rats in an object recognition task: Rats that were given an acute dose of

nicotine demonstrated improved working memory compared to normal controls due to a

clear increase in the recognition of an old object vs. a new object (Puma, Deschaux,

Molimard, & Bizot, 1999). In addition to this, zebrafish that were exposed to an acute

dose of nicotine for 3 minutes showed increased performance in a spatial position

discrimination task (Levin, Limpuangthip et al., 2006).

One way to demonstrate working memory is to expose rats to the Radial Arm

Maze (RAM), which consists of a maze with arms extending out from a center (the

number of arms varies based on the study), and a reward at the end of each arm (usually a

food treat). Rats should only venture down each arm once until all rewards are received,

and any time a rat re-enters an arm, it is marked as an error. In this way, working

memory can be assessed by the extent to which the rat remembers what arms it has

already been down. In a study assessing the effects of acute and chronic nicotine on

young and aged rats in the RAM, Levin and Torry (1996) found that the young rats

treated with chronic nicotine showed an increase in working memory, but the aged rats

did not show the same increase. There was no significant improvement in working


memory after acute doses of nicotine in the young rats, but there was a pronounced effect

of the acute administration of nicotine in the aged group (Levin & Torry, 1996). These

findings suggest that a chronic dose of nicotine may be more effective in cognitive

enhancement in young rats, while aged rats may benefit more from acute doses

administered immediately prior to a task (Levin & Torry, 1996). These findings contrast

slightly with the findings of French and colleagues (2006), who tested aged rats with a

chronic nicotine dose of 0.3 mg/kg in the RAM. They found nicotine significantly

increased working memory in these rats, and suggested that the lower dose in aged rats

helped improve performance due to their decreased metabolism of the nicotine (French,

Granholm, Moore, Nelson, & Bimonte-Nelson, 2006). In the Levin and Torry (1996)

study, the dose given to both groups was 0.5 mg/kg. It is possible that this dose was

effective for the young rats, but too much for the old rats when administered chronically.

Another commonly used method to demonstrate increased cognition in rats

exposed to nicotine is to test them in the Morris water maze (MWM) to determine their

cognitive spatial abilities. The MWM is a large circular maze filled with opaque water,

and rats (or mice) must locate a hidden platform using spatial cues surrounding the maze

(Morris, 1984). At the start, rats are placed on the platform so they can become

acclimated to the maze and exposed to the spatial cues that surround it. Then they must

learn that there is no escape from the maze via the outside walls, and they must utilize the

spatial cues around the maze to locate the platform. Full acquisition of the task occurs

when the swim times decrease and level off, and remain low when the rats are placed at

varying start locations around the maze (Morris, 1984). Studies have demonstrated that

rats differ in performance in the Morris water maze based on age, gender, and sometimes


strain (Hansalik, Skalicky, & Viidik, 2006; Topic et al., 2005). Age differences are

apparent in that aged rats tend to have more difficulty in the acquisition of the water maze

task compared to young rats, however according to a study by Topic and colleagues

(2005), there is considerable variability due to individual differences. Thus, while most

aged rats were slower than the young rats, there were some aged rats who had swim times

that were on par with those of the youngest and fastest rats (Topic et al., 2005). Early

training, however, can decrease response times of aged rats compared to aged rats with

no previous training, suggesting that the MWM is also a tool for assessing long-term

memory retention (Hansalik et al., 2006).

In nicotine studies examining spatial learning, the dose administered and length of

administration prior to training are extremely important. A study using mice

demonstrated that a nicotine dose of 0.7 mg/kg/day produced significant impairment in a

water maze task, but 0.35 mg/kg/day administered intraperitoneally 5 days prior to

training and throughout training itself produced significantly lower trial latencies (Bernal,

Vicens, Carrasco, & Redolat, 1999). Thus, it is important not only to administer a low

dose of nicotine, but also to administer it a few days prior to the task to allow the drug to

take its full effect (Bernal et al., 1999). The extra time needed is most likely due to the

fact that receptor upregulation, which can enhance the effect the nicotine has on the brain,

takes some time to occur. Nicotine doses as low as 0.2 mg/kg have been shown to have

an effect on both young and older rats, and these studies have also utilized the technique

of waiting a few days prior to the first acquisition trials in order for the drug to take effect

(Socci, Sanberg, & Arendash, 1995). Using a dose of 0.2 mg/kg not only results in

enhanced water maze acquisition in aged rats, but it also has been shown to improve


memory retention in young rats (Socci et al., 1995).

It has also been reported that high doses of nicotine do not demonstrate nicotine’s

positive effects due to ACh receptor desensitization. Receptor desensitization occurs

when a neurotransmitter binds to a receptor but the ion channel, through which positively

charged ions would normally travel to lead to an EPSP, remains closed (Benwell et al.,

1995). Thus, though the neurotransmitter is bound to the receptor, an action potential

does not occur and the neuron does not fire. Because of this desensitization (which

occurs when rats are administered high levels of nicotine that are on par with the amount

a regular smoker may receive, 1.0 and 4.0 mg/kg/day for this particular study), the effect

of the nicotine on memory and learning is actually inhibitory (Benwell et al., 1995).

Lower doses of nicotine, for example, 0.25 mg/kg/day, did not show this desensitization,

and thus still shows cognitive enhancement in spatial learning studies (Benwell et al.,

1995).

Long-Term Potentiation

Studies involving nicotine treatment in working memory tasks are also beneficial

in that they can provide evidence for nicotine’s inducement of Long Term Potentiation

(LTP). As was mentioned earlier, LTP occurs in the hippocampus and results in an

increase in synaptic firing and effectiveness (Schumann et al., 2004). LTP is an

important part of memory formation, and it has been shown to occur throughout the

neuronal information pathways in the brain (Schumann et al., 2004). This process also

involves the upregulation of glutamate NMDA receptors, which have been shown to be

modulated by nicotine in some studies of working memory in rats (Levin, Bettegowda,

Weaver, & Christopher, 1998).


One study looked at nicotine’s effect on LTP in the rat hippocampus by assessing

its therapeutic effects in rats suffering from working memory impairments. The working

memory impairments were induced by stress, and the subcutaneous administration of 1

mg/kg of nicotine prevented the memory impairments associated with the stressor

(Aleisa, Alzoubi, Gerges, & Alkadhi, 2006). The nicotine served to normalize the LTP

that had been reduced in the stressed rats treated with saline. This suggests that nicotine

does have neuroprotective effects regarding memory and learning, so much so that it can

counteract the impact of a stressor in rats (Aleisa et al., 2006). The control rats in the

study that were not exposed to the stressor but received nicotine treatment did not show

any learning or memory improvement, however this could be due to the extremely high

dose (1 mg/kg) of nicotine that was administered. It has already been demonstrated that

high doses can show water maze impairment, so it is possible that such a high dose is

only necessary to reverse the effects of cognitive impairment, not to enhance the

performance of otherwise normal rats (Bernal et al., 1999).

It has also been demonstrated that nicotine can attenuate working memory

impairments caused by dizocilpine, an NMDA glutamate receptor antagonist, providing

more evidence for nicotine’s neuroprotective effects (Levin, Bettegowda et al., 1998).

NMDA glutamate receptors are known to be upregulated as a result of LTP, so the

attenuation of these impairments could be due to an increase of LTP as induced by the

nicotine. Because the nicotine stimulated the release of glutamate, it was able to

counteract the drug and reverse the working memory impairments, though it did not

increase performance in the radial arm maze. When the dose of nicotine (0.4 mg/kg) was

given without the dizocilpine, an increase in maze performance was also not detected—it


is possible that this dose was too high to produce positive effects, as it has been

demonstrated that a dose of 0.7 mg/kg in mice actually causes impairments in spatial

learning tasks (Bernal et al., 1999; Levin, Bettegowda et al., 1998). In a later study

involving dizocilpine and nicotine, the nicotine dose of 0.4 mg/kg led to inconclusive

results regarding its neuroprotective effects. This suggests that 0.4 mg/kg may be at

some kind of dose threshold where the effects of the nicotine switch from positive to

negative (Levin et al., 2003).

Neurogenesis

In past studies demonstrating nicotine’s attenuation of working memory

impairments, a case has been made that nicotine can be neuroprotective, meaning it

minimizes the damage done by certain drugs or events by stimulating the release of

neurotransmitters such as glutamate or dopamine (Levin, Bettegowda et al., 1998).

Despite this, a study has indicated that nicotine actually decreases neurogenesis in the

brain when self-administered by rats (Abrous et al., 2002). However, in this study, the

self-administration aspect led rats to have plasma nicotine levels that were extremely high

and equivalent to those of smokers—the rats in cognitive enhancement studies as well as

humans in controlled nicotine patch studies do not receive such high levels of nicotine

(Abrous et al., 2002). It has already been reported that such high nicotine levels

desensitize important receptors involved with the release of both glutamate and

dopamine, neurotransmitters essential to memory and learning (Benwell et al., 1995).

For this reason, it makes sense that large amounts of nicotine may actually decrease

neurogenesis in the brain.

Studies employing the use of bromodeoxyuridine (BrdU) are essential in


demonstrating the effects of spatial learning tasks on neurogenesis, and can indicate

whether nicotine has any effect on the cell proliferation. When injected intraperitoneally,

BrdU incorporates into the DNA of all cells undergoing synthesis at the time of learning.

The BrdU stays in these cells and can be detected post-mortem through

immunohistochemical procedures (Cooper-Kuhn & Kuhn, 2002). It has been

demonstrated that an increase in newly-generated cells in the hippocampus occurs due to

the process of learning itself—not just training (Dalla, Bangasser, Edgecomb, & Shors,

2007). Even when the task was not hippocampus dependent, rats that “learned well,” that

is, that demonstrated a high acquisition of the task after its completion, showed increased

cell proliferation compared to rats that did not learn well, thus emphasizing the

importance of learning rather than training (Dalla et al., 2007). BrdU studies have also

clearly shown that neurogenesis decreases significantly from adolescence to adulthood,

though neurogenesis in the hippocampus is still apparent in adults as a result of certain

tasks involving learning (He & Crews, 2007). Though the cell growth seen as a result of

the Morris water maze task is generally attributed to the learning that occurs during the

late phase of this task, there is also evidence that exercise can increase cell proliferation

as well (van Praag, Kempermann, & Gage, 1999). This suggests that tasks involving

exercise, such as the Morris water maze, could be even more indicative of cell

proliferation in the hippocampus.

Morris water maze studies involving BrdU injections can provide useful results

regarding neurogenesis in the hippocampus as a result of the learning that occurs during

the task. In one Morris water maze BrdU study, the early phase of learning, in which

there is a rapid improvement in performance, did not produce significant cell


proliferation, while the late phase of learning, during which the highest point of

performance is reached, demonstrated a significant increase in the number of newly

generated cells (Dobrossy et al., 2003). This was thought to potentially be due to the

stress of the early phase of learning, in which rats were becoming acclimated to the

task—for this reason, perhaps, neurogenesis was dampened at that time (Dobrossy et al.,

2003). Neurogenesis in the late phase, it was hypothesized, could indicate that the

dentate gyrus of the hippocampus was available for the integration of new information

and the storage of new memories at that phase of learning (Dobrossy et al., 2003).

Studies using lower doses of nicotine in tasks that are known to produce

neurogenesis in the brain, particularly in the hippocampus, have shown that low doses of

nicotine do not have a detrimental effect on neurogenesis (Scerri, Stewart, Breen, &

Balfour, 2006). In accordance with the findings of previous studies, Scerri and

colleagues (2006) concluded that a nicotine dose of 0.4 mg/kg per day had an adverse

effect on neurogenesis (as demonstrated by a BrdU investigation), but rats receiving 0.25

mg/kg per day showed an increase in cell proliferation in response to the Morris water

maze task. This study provides further evidence that learning associated with the Morris

water maze task can be demonstrated clearly in the dentate gyrus of the hippocampus,

and that nicotine may have a positive impact on the cell proliferation that occurs there

(Scerri et al., 2006).

c-Fos Expression

In addition to increased cell proliferation, it has been widely reported that nicotine

produces an increase in c-Fos expression in various areas of the brain (Guan, Kramer,

Bellinger, Wellman, & Kramer, 2004; McCormick & Ibrahim, 2007; Panagis, Nisell,


Nomikos, Chergui, & Svensson, 1996; Salminen, Lahtinen, & Ahtee, 1996). c-Fos is a

protein that indicates neuronal activation, and it has often been studied in relation to

stress (Kovacs, 1998; McCormick & Ibrahim, 2007). In a study combining stressors with

nicotine treatment in adolescent male and female rats, it was found that males showed an

increase in c-Fos in the hypothalamus while females did not, and chronic nicotine groups

showed more c-Fos expression than did acute nicotine groups (McCormick & Ibrahim,

2007). From these results, it can be hypothesized that nicotine must be chronically

present in the system in order to have an effect on c-Fos expression in the brain. It has

also been found that nicotine significantly increases c-Fos expression in brain areas

specifically related to stress, such as the paraventricular hypothalamic nucleus and the

supraoptic nucleus (Salminen et al., 1996).

The rate of nicotine delivery can also have an effect on c-Fos expression (Samaha

et al., 2005). In one study, rats were infused with either 25 or 50 µg/kg nicotine in a time

span of 5, 25, or 100 seconds. The results showed that c-Fos expression and receptor

sensitization were highest when the infusion rates were fastest. High administration rates

are thought to be central to the addictive properties of nicotine, and perhaps the increase

in c-Fos expression is a result of the rapidly increasing occupancy of nicotinic receptors

that occurs when nicotine is delivered at a fast rate (Samaha et al., 2005).

Nicotine can also cause Fos increases in areas of the brain specifically related to

dopamine transmission, such as the nucleus accumbens and the caudate-putamen. In a

study looking at both c-Fos and ∆FosB (another member of the Fos family that stays in

the brain longer and has been linked to drug addiction), nicotine was shown to increase

both Fos types in these dopaminergic brain areas, providing further support for nicotine’s


role in increasing dopamine transmission (Marttila, Raattamaa, & Ahtee, 2006). The

increase that nicotine causes in locomotor activity, also known as behavioral

sensitization, has also been related to an increase in Fos in the nucleus accumbens, the

striatum, and the ventral tegmental area, again providing support for nicotine’s role in the

dopamine release process, and providing further evidence for its rewarding properties

(Shim et al., 2001).

Increased c-Fos expression in nicotine studies is also dependent on the nicotine

dose used. One study found that a nicotine dose of 2 mg/kg showed the most dramatic

expression of c-Fos in the brain, while the lower doses of 0.5 mg/kg and 1mg/kg showed

minimal increase in c-Fos (though these doses did still produce an increase when

compared to normal controls) (Ren & Sagar, 1992). Other studies have shown an

increase in c-Fos expression in their brain areas of interest (for example, the

hypothalamus, the visual pathways, the striatum) using doses of 0.4 mg/kg, (Shim et al.,

2001), 0.5 mg/kg (McCormick & Ibrahim, 2007), and 1mg/kg (Salminen et al., 1996).

Though these doses are slightly higher than those used in spatial learning studies, the fact

that they produce c-Fos expression suggests that lower doses may still lead to an increase

in expression when compared to normal controls.

The fact that chronic nicotine shows an increase in c-Fos in certain brain areas

provides support for the idea that neurons may undergo changes in gene expression due

to nicotine exposure (Ren & Sagar, 1992). This alteration of neuronal gene expression,

which could potentially be long-term, would be beneficial to people suffering from

diseases that affect cognition because it could mean an increase in nAChrs, which would

increase their cognition and potentially delay the onset of cognitive decline (Ren &


Sagar, 1992).

The purpose of the current study was to assess the effects of a chronic dose of

nicotine on the ability of rats to acquire the Morris water maze task in adolescence and

adulthood. Previous studies of chronic nicotine administration have indicated that doses

around 0.2 mg/kg serve to increase cognitive functioning, and that higher doses run the

risk of adversely affecting maze performance (Bernal et al., 1999; French et al., 2006;

Socci et al., 1995). For this reason, we used 0.2 mg/kg as our dose for rats of all age

groups.

Because most of the previous studies involve specifically young or aged rats, this

study was unique in that it examined the effects of nicotine on the same rats in

adolescence (Phase 1) and adulthood (Phase 2). The schedule of nicotine was also varied

based on the groups. There were four main groups: rats that received nicotine in Phase 1

during adolescence and nicotine again in Phase 2 when they reached adulthood (N/N),

rats that received nicotine in Phase 1 and saline in Phase 2 (N/S), rats that received saline

in Phase 1 and nicotine in Phase 2 (S/N), and rats that received saline in Phases 1 and 2

(S/S). In Phase 1, rats were trained in the Morris water maze while receiving either 0.2

mg/kg of nicotine or saline for 2 weeks, and then nicotine or saline administration was

ceased until four months later. At this time, drug treatment was resumed based on the

group, and Morris water maze testing was repeated in the same fashion. Additionally,

Bromodeoxyuridine (BrdU) injections were introduced into the study during the last two

days of Phase 2 acquisition in order to view possible neurogenesis as a result of the

nicotine exposure and water maze training (Scerri et al., 2006).

Though this study involved nicotine cessation after a 2-week period of chronic


nicotine administration, the rats were not expected to experience serious withdrawal

symptoms. A study investigating withdrawal from chronic nicotine showed that even

after an extremely high dose (22.2 mg), withdrawal symptoms in adolescent rats were

minimal, even after the immediate introduction of a nicotinic agonist (Wilmouth & Spear,

2006). Adult rats showed minor withdrawal symptoms that abated shortly after nicotine

cessation (Wilmouth & Spear, 2006). This finding, coupled with the fact that the dose

was significantly higher in these studies, suggests that the rats in the present study were

not in danger of severe withdrawal symptoms in the interim between phases.

The purpose of the varying nicotine schedules was to determine whether learning

was state dependent (for the N/N group), whether early experience had a greater effect on

task acquisition (for the N/S group), or whether the effect of nicotine was more

pronounced in older rats (for the S/N group). The rats that received saline in both phases

served as a control for this experiment. It was expected that, in both phases, nicotine rats

would perform better than saline rats in the Morris water maze. Due to the previous

training that the rats received in Phase 1, it was expected that reaction times would be

faster overall in Phase 2 during the re-acquisition of the task, however it was

hypothesized that rats receiving nicotine in Phase 2 would re-acquire the task faster than

rats not receiving nicotine, and that rats receiving nicotine in both phases would be better

at the task overall (Hansalik et al., 2006).

It was also expected that rats in all groups would show an increase in cell

proliferation in the hippocampus due to the water maze task, and that the Phase 2 nicotine

rats would have more BrdU expression than the saline rats due to previous studies

suggesting nicotine is neuroprotective and involves nAChR upregulation (Dobrossy et


al., 2003; Wang et al., 2008). We also tested for c-Fos in the hippocampus following the

last day of training in the second phase of testing for all rats. Based on previous studies

indicating that chronic nicotine at doses of 0.5 mg/kg to 2 mg/kg increase c-Fos

expression in certain brain areas, we expected c-Fos levels to be elevated in rats receiving

nicotine in Phase 2 (McCormick & Ibrahim, 2007).

We specifically looked at c-Fos expression in the dentate gyrus of the

hippocampus. A previous study examining nicotine’s effect on c-Fos in the pigeon brain

reported the hippocampus to be one of the greatest sites of c-Fos expression, however to

our knowledge a study investigating c-Fos expression in the rat hippocampus following

nicotine exposure and a spatial learning task has not previously been attempted

(Chadman, Woods, & Stitzel, 2007). For this reason, the present research provides

important results regarding the effect of nicotine on c-Fos in the hippocampus and the

implications this has regarding spatial learning. A significant c-Fos increase in the

hippocampus may provide even further evidence that nicotine has a significant effect on

cell growth and receptor upregulation in this brain area, as c-Fos is generally an indicator

of the beginnings of neuronal change and activity (McCormick & Ibrahim, 2007; Ren &

Sagar, 1992).

Method

Subjects

Subjects included 50 male Fisher-344 rats born at the Charles River Laboratories

in Stoneridge, NY, USA. Rats from all cohorts were 6 weeks old on arrival. The study

began 2 weeks later, at which time rats in Cohort 1 (numbers 1-16) weighed between 160

g and 185 g, rats in Cohort 2 (numbers 17-35) weighed between 155 g and 180 g, and rats


in Cohort 3 (numbers 36-50) weighed between 150 g and 180 g. Five months later

during Phase 2 of the experiment, Cohort 1 weighed between 305 g and 340 g, and

Cohort 2 weighed between 300 g and 345 g, with one rat weighing 276 g.

Rats were housed 3 or 4 to a clear plastic cage measuring 19 x 9 x 9 in. They

were given regular shavings, water, and rat chow. Some of the rats from Cohorts 1 and 2

were given wet food along with the dry food a few months into the study due to slightly

decreased weight that may have been the result of a mouth infection. Rats were also

given a square red Plexiglas tube to use as a shelter.

Procedures were approved by the Connecticut College Animal Care and Use

Committee in accordance with the guidelines of the USDA and the Office of Laboratory

Animal Welfare.

Apparatus

Alzet osmotic mini-pumps (model 2004, Braintree Scientific, Braintree, MA)

were used to administer a chronic dose of 0.2 mg/kg of nicotine or saline per day for a 2-

week period.

Activity levels were recorded in an activity box measuring 42 x 45 cm on the

inside and 11 cm high, with a normal rat cage, measuring 42 x 22 cm and 21 cm high,

inside of it. The normal cage was made of transparent plastic and had a metal lid and

wood shavings for bedding. The activity box surrounding the cage was made of metal

and measured the activity of the rat. There was a light beam on one side of the cage

projecting to the other side, which measured beam crossings (the amount of times the rat

interrupted the light). This provided a quantitative measurement of the rat’s movements.


Spatial learning was tested using a Morris water maze. The maze was a large

white plastic tub that was 4 ft in diameter. The water level was kept at 13 in, with the

platform approximately 0.5 in below the water level. The platform was a tall Plexiglas

tube with a petri dish fitted to the top. A white washcloth was attached to the top of the

platform with a rubber band. The platform was kept stationary, 11 in from the right side

of the maze, using a weight at the base. The water was made opaque with white tempera

paint, which was mixed thoroughly in the water prior to each acquisition and test day.

The water was kept at 20 °C during all experimental trials.

The maze was in a rectangular shaped room with white walls and various

stationary visual cues surrounding it. The visual cues included rectangular pieces of

construction paper with different shapes (circles, a star, stripes) of contrasting color on

top of them. The metal temperature gauge and hose, as well as the open door and the

experimenter’s position also acted as visual cues, and remained constant during the

experiment. The maze was 10.5 in away from the two longer walls, and 17 in away from

the shorter wall farthest from the door to the room.

Procedure

Rats were allowed to acclimate to their new surroundings for one week prior to

being handled and weighed for the first time. The rats were handled for approximately 3

minutes each on at least two separate occasions prior to their first day of surgery. Weight

measurements (in grams) were taken two days prior to surgery, as well as the day of

surgery in all cases, in order to ensure the rats were gaining weight at a normal pace and

to make the measurement of the necessary anesthetic as accurate as possible. Activity

levels were taken on the day of the last surgery for all groups in order to determine


possible changes in locomotion in the nicotine rats. The early phase of acquisition trials

in which Cohorts 1 and 2 (these two cohorts make up what is later referred to as the dual-

phase cohort) were first exposed to the experimental procedure was considered Phase 1.

Cohort 1 initially contained 16 rats, however due to some complications with the pumps

the data from 2 of the rats was not used. Cohort 2, containing rats that arrived one week

after Cohort 1 with all of the same specifications, included 18 rats. One rat died during

the surgical procedure, so its data was not used in the experiment. At this time, rats were

given either nicotine or saline for the experimental procedure. See Table 1 for a

description of the drug treatments and number of subjects in each group.

During Phase 2 of testing, rats in the dual-phase cohort were broken up into four

groups, N/N, N/S, S/N, and S/S, depending on what drug treatment they received in

Phases 1 and 2 (see Table 1). The purpose of the N/N rats was to indicate whether

acquisition of the procedure was state-dependent. The N/S rats were to demonstrate

whether early experience had a greater effect on task acquisition than the nicotine itself.

The S/N rats were to demonstrate whether the effect of the nicotine would be more

pronounced in older rats, and the S/S group acted as a control for the three other groups.

The 16 rats in Cohort 3 (the single-phase cohort) contained the same

specifications as all previous rats, and underwent the same procedure, with 8 receiving

nicotine pumps and 8 receiving saline pumps. One rat died during the procedure, leaving

7 nicotine rats and 8 saline rats. After one phase of acquisition and test trials, these

animals were sacrificed and their brains were examined for neurogenesis (as evidenced

by BrdU) and c-Fos in the hippocampus. In this way they acted as a control for age, as

their results were expected to provide insight into the c-Fos and BrdU expression that


might occur in adolescent rats as a result of nicotine exposure. They did not undergo a

second testing phase.

Approximately 24 hours prior to the surgery the pumps were set up following the

procedure as outlined by the Alzet company. The nicotine was mixed using 0.2859

grams of nicotine per 3 ml of saline. This concentration allowed the animals to receive

0.2 mg/kg per .29 µl nicotine base per day, while the control animals would receive an

equal volume of vehicle (saline) per day. In order to fill the pumps, the empty pump was

first weighed along with the flow moderator. Solution was drawn into a 1 ml syringe and

a 27 gauge filling tube (provided by the Alzet company) was fit to the end of it. The

empty pump was held upright and filled slowly, ensuring there were no air bubbles in the

pump. When the solution appeared at the outlet of the pump, the filling tube was

removed and excess solution was wiped off. The flow moderator was then inserted until

it was flush with the pump, and the displaced solution was wiped off. The pump was

then weighed again, ensuring that the difference in the weight from the empty pump gave

the proper net solution of the pump, 30 μl. Pumps were then incubated in sterile saline

and allowed to equilibrate at 37 °C overnight.

On the day of surgery, all animals were weighed in order to ensure an accurate

dosage of anesthetic. The anesthetic was prepared by mixing 1.95 ml of xylazine and 1.8

ml of ketamine. In order to determine the specific amount of anesthetic necessary for

each rat, 1.25 ml was multiplied by the most recent weight measurement in kilograms.

The result, usually hovering around 0.2 ml, was administered intraperitoneally and the

rats were put in a covered cage as the drug took effect. Once the rat was asleep, a small

area between the shoulder blades was shaved, and a vertical incision was made in the


skin. The pump was then inserted subcutaneously. The wound was then closed with two

metal clips, and the rat was placed in another covered cage until the anesthetic wore off.

This procedure was repeated for each rat.

During the first surgeries for Cohort 1, the pumps were inserted with the top of

the pump facing the wound. Due to the complications of a few of the pumps coming out

because of excessive scratching, it was determined that the release of nicotine may have

been irritating the wound. Thus, all other surgeries were conducted by inserting the top

of the pump first.

Two days after the surgery all rats were weighed to ensure they did not lose a

significant amount of weight as a result of the drug. The rats were given 7 days prior to

the acquisition trials for the drug to begin to take effect. After this time had elapsed, all

rats were tested in the Morris water maze.

Testing began daily at 4:00 pm, and usually lasted between 1.25 and 2 hours.

Prior to the first trial on Day 1 of acquisition, all rats were placed on the platform for 60

seconds so they could become acclimated to their surroundings. During the acquisition

trials, rats were positioned at start location 1 (see Figure 1) and a time measurement was

taken with a stopwatch to measure how long it took them to reach the platform. If any rat

failed to reach the platform after 60 seconds of swimming during any given trial, the rat

was removed from the maze and placed on the platform for another 60 seconds.

Acquisition involved five trials from start location 1 per day for five consecutive

days. Rats were tested by cage, so trials were alternated between 4 rats at a time in order

to avoid fatigue from completing multiple trials per rat in succession. After the fifth day

of acquisition trials there were two days without any trials, followed by one day of test


trials (see Table 2). During the testing day, the first trial was at the familiar start location

(start location 1), and the remaining 4 trials began at varying start locations around the

maze (see Figure 1 for a diagram of the maze and start locations). These start locations

remained constant throughout the study.

It should be noted that during the first day of acquisition trials for Cohort 1 in

Phase 1 of testing, 10 acquisition trials were taken per rat (as opposed to 5). It was

determined that this number was too high, as latencies began to increase toward the end

of the acquisition period. The increase was most likely due to fatigue, and thus all

acquisition days following that first day involved only five trials, and only the first five

trials from acquisition day 1 for Cohort 1 were used in the data analysis.

After the week of testing was over, rats were weighed, activity levels were

recorded in an activity box for five minutes, and then they were anesthetized using the

same procedure as outlined previously. The pumps were removed and the rats from the

dual-phase cohort were kept until Phase 2 of testing, which occurred four months later.

These rats were weighed once a week in between testing phases.

Acquisition and testing for the single-phase cohort and Phase 2 of the dual-phase

cohort were subject to a slightly different procedure. Surgery was performed on these

rats as per usual, and acquisition trials began one week following the procedure. The

procedure for acquisition trial days 1-4 were identical to those of Phase 1 of the dual-

phase cohort, however after the fourth day one day was skipped, and the last day of

acquisition was on the sixth day of the experiment. The test trials were on the day

immediately following the last day of acquisition (day 7), in order to allow for perfusion

of rats that coincided with the test trials and the injection of BrdU (see Table 2 for the full


experimental schedule). This change in procedure was due to complications with the

BrdU, however it was not expected that any significant difference would arise due to this

change. The order in which the trials occurred and the small timing changes of the

testing trials were not expected to have any effect on the ability of the rats to perform the

task and to recall it during testing.

BrdU was introduced during Phase 2 acquisition trials for the dual-phase cohort,

and during the acquisition trials of the single-phase cohort. BrdU was mixed in a

concentration of 25 mg/ml in physiological saline. It was necessary to warm the solution

in order for the BrdU to dissolve. Once the BrdU was in solution, rats were weighed, the

proper amount for injection (2 ml/kg) was calculated, and rats were injected at roughly

4:30 pm during the free day between acquisition days 4 and 5. The second BrdU

injection occurred 30 minutes prior to acquisition trials on acquisition day 5, which was

one day prior to test trials. Two rats from the dual-phase cohort did not receive the BrdU

injection, as they were aggressive and difficult to inject.

Rats were perfused 90 minutes after their last test trial in order to preserve the

brains to look at c-Fos and incorporation of BrdU in the hippocampus. This process

involved euthanizing the rat and perfusing the body with the fixative 4% PFA, then

removing the brain and soaking it in the fixative overnight. The following day, the brain

tissue was transferred to a 30% sucrose mixture. Using a cryostat, the brain tissue for

each rat was then frozen and sliced at 25μm through the dentate gyrus of the

hippocampus. The sections of brain tissue were then placed in a preserving solution, and

3 slices from each rat were selected to be stained and examined further for c-Fos content.

A separate set of sections was selected to be stained for BrdU incorporation. The


sections of tissue were chosen based on their representation of the dentate gyrus. A rat

brain atlas was utilized to determine which sections of the brain tissue were to be

examined (Paxinos & Watson, 1998). One rostral (AP –3.30 mm in reference to

Bregma), middle (AP –2.12 mm in reference to Bregma) and caudal (AP –3.6 mm in

reference to Bregma) section was taken for each rat to represent different sections of the

hippocampus (Paxinos & Watson, 1998).

c-Fos Immunohistochemistry

The c-Fos staining procedure involved washing the selected pieces of tissue for 30

minutes and the placing them in a solution containing the 10 antibody (rabbit anti-Fos

polyclonal IgG, Santa Cruz Lot #c270) that binds to segments of the c-Fos protein. The

concentration was 1:8000, diluted in 50 ml of blocking solution (98.75mL 0.01 M PBS, 1

ml normal goat serum, 1 g bovine serum albumin, 0.25 ml 30% Triton X100). They were

left in this solution overnight, and then rinsed and placed in a solution containing

biotinylated 20 antibodies (goat anti-rabbit polyclonal IgG, Jackson Laboratories) that

recognize the 10 antibody. This concentration was 1:200 diluted in 50 ml of blocking

solution. Two hours later the tissue was rinsed again and avidin-biotin molecules

complexed with horseradish peroxidase diluted in 50 ml of 0.01 M PBS were added.

These molecules bind to the biotin molecules on the antibodies. After one hour enhanced

DAB (Diaminobenzidine enhanced with nickel and cobalt) diluted in 50 ml of 0.1 M PB

was added, which interacted with the peroxidase to form a precipitate that allows for the

visualization of the c-Fos location.

After being left to dry overnight, the slides were coverslipped and viewed under a

microscope. A 240 x 152 µm portion of the dentate gyrus was viewed in each section of


tissue for each rat. The dimensions of this area were kept constant across rats. For the

middle and caudal sections, the portion of the dentate gyrus that was measured for c-Fos

content was the hilus. For the rostral sections, the lower portion of the dentate gyrus was

measured, and this location was kept constant across caudal sections. Only the darkest

particles in each section were counted, ensuring that only Fos-positive nuclei were being

measured.

BrdU Immunohistochemistry

The BrdU staining procedure was performed as outlined in the kit from Chemicon

International. The procedure first involved the deparaffinization of the control tissue

included with the kit. This involved soaking the paraffinated slides in xylene, and then

washing them in 100%, 90%, 80%, and 70% ethyl alcohol for 3 minutes each.

Afterwards tissue was washed in PBS for 3 minutes. Control slides and experimental

slides were soaked in Quenching Solution (dilute 30% hydrogen peroxide 1:10 in

methanol) for 10 minutes, and then washed with PBS for 2 minutes. Two drops of 0.2%

Trypsin Solution were added to control slides and slides were incubated at room

temperature for 10 minutes. Slides were then washed for 3 minutes in distilled water.

Denaturing Solution was added to all slides and they were allowed to incubate at room

temperature for 30 minutes, followed by 2 PBS washes for 2 minutes each. Blocking

solution was then added to each slide, and they incubated for 10 minutes. Slides were

blotted dry on a paper towel before the Detector Antibody was added, and then were left

to incubate for 60 minutes. Following incubation slides were washed twice with PBS, 2

minutes per wash.

Streptavidin-HRP Conjugate was then added to each slide and slides were


incubated at room temperature for 10 minutes. This was followed by two 2-minute

washes in PBS. DAB concentrate was added to slides at a concentration of 1 µl for every

29 µl of Substrate Reaction Buffer. Slides were allowed to incubate for 10 minutes and

were washed with distilled water for 2 minutes. Hematoxylin Counterstain was added to

all slides and left to incubate for less than a minute. Slides were briefly washed with

distilled water and then incubated in PBS for 1 minute until the color turned blue. Slides

were washed for 2 minutes in distilled water. To prepare for coverslipping, slides were

incubated in 90% ethanol for 30 seconds, 100% ethanol for 30 seconds, and xylene for 30

seconds. Slides were coverslipped and left to dry overnight.

Results

Weight/activity level analysis

Weights and activity levels were monitored throughout the study to determine if

the administration of chronic nicotine led to significant weight loss or increased

locomotion. One-way ANOVAs were conducted for the weights of the dual-phase cohort

after they had been exposed to nicotine or saline for 14 days in Phase 1 and in Phase 2.

The results for these rats were not significant, indicating that after 14 days of nicotine

administration in both phases, no significant differences arose between the weights of the

saline and nicotine rats. The single-phase cohort also did not demonstrate significant

differences between the weights of rats receiving nicotine and rats receiving saline.

One-way ANOVAs were also conducted for the activity levels of the dual-phase

cohort (in Phases 1 and 2) and the single-phase cohort after they received nicotine or

saline for 14 days to view potential increases in locomotion as a result of nicotine

administration. No significant results were found between the activity levels of the


nicotine or saline rats in any cohort. This shows that the nicotine did not have a

significant effect on the locomotion of the rats during the experiment.

Spatial learning analysis

Single-phase cohort

A repeated measures analysis using a 2 (group) x 5 (days) mixed design ANOVA

was conducted with trial totals across the 5 days of acquisition. Trial totals were

calculated by adding the latencies of trials 1 through 5 for each rat in each day. The

independent variable was the drug condition and the dependent variables were the trial

latencies.

The effect of condition across days using trial totals was not significant,

indicating that condition had no significant effect on trial totals across days 1 through 5.

However, an effect of training could be found by comparing the trial latencies across

days for all rats, regardless of drug condition. In the analysis of trial totals, a significant

effect of training was seen across the days of the acquisition period, F(4, 52) = 21.49, p <

.001. This shows that the rats learned the task due to an overall decrease in trial latencies.

Performance between groups on each acquisition day was assessed using a 2

(group) x 5 (trials) mixed design analysis of variance (ANOVA), which was conducted

with repeated measures across the 5 trials per day. The effect of condition (nicotine or

saline) on trial latencies for each individual day was not significant, indicating no

significant difference between rats treated with nicotine and rats treated with saline. In

the analyses of individual days, however, a significant effect of training was found for

acquisition days 1 (F(4, 52) = 4.18, p = .005), and 3 (F(4, 52) = 3.18, p = .02), and effects

for days 2, 4, and 5 were not significant (see Figure 2 for a graph of mean trial latencies


in acquisition days 1-5 that demonstrate the learning of the task). This indicates that the

majority of learning occurred across trials on days 1 and 3, during the earlier days of

acquisition.

A one-way ANOVA was conducted for test trial totals (the sum of all test trials

for each rat), and a separate ANOVA was conducted for test trial averages (the sum of all

test trials for each rat divided by the number of trials). The effect of condition for the

trial totals and averages were not significant, however the means for saline (M = 30.02)

and nicotine (M = 26.52) rats indicate a trend toward significance (see Figure 3 for a

graph of test trial averages at each start location). Additionally, five one-way ANOVAs

were conducted for each test trial (each test trial was at a different start location, and

there were 5 in total) in order to view possible effects of condition. Effects of condition

for test trials 1 through 5 were not significant, indicating that the nicotine did not produce

a significant decrease in trial latencies in adolescent rats.

Dual-phase cohort

Phase 1

Statistical analyses for Phase 1 acquisition were conducted using the same

procedure as outlined for the single-phase cohort. A repeated measures analysis using a 2

(group) x 5 (days) mixed design ANOVA was conducted with trial totals across the 5

days of acquisition. Trial totals were calculated by adding the latencies of trials 1

through 5 for each rat in each day. Again, the independent variable was the drug

condition and the dependent variables were the trial latencies.

The results for this group of adolescent rats in acquisition coincided with those of

the single-phase cohort adolescent rats. The effect of condition across days using trial


totals was not significant, confirming that nicotine did not have a significant effect on

trial totals across acquisition days 1 through 5. The effect of training for trial totals

across days 1 through 5 was significant, F(4, 116) = 57.54, p < .001, indicating learning

due to a decrease in latency totals across acquisition days in Phase 1 acquisition.

Performance between groups on each acquisition day was assessed using a 2

(group) x 5 (trials) mixed design ANOVA, which was conducted with repeated measures

across the 5 trials per day. The effect of condition (nicotine or saline) on trial latencies

for individual days 1, 2, 4, and 5 were not significant, indicating the nicotine did not have

a significant effect in the adolescent rats in Phase 1 of acquisition. There was a

significant effect of condition seen for Day 3, however, indicating a decrease in trial

latencies in the nicotine treated rats, F(4, 116) = 2.56, p = .04. Additionally, there was a

significant effect of training in day 1, F(4, 116) = 7.22, p < .001, and day 2, F(4, 116) =

2.95, p = .02, indicating learning via a decrease in latencies across trials in both

conditions. The effects of training on days 3, 4, and 5 were not significant, coinciding

with the results from the single-phase cohort suggesting that the bulk of learning occurs

during the early days of acquisition (see Figure 4 for a graph of mean trial latencies in

Phase 1 acquisition days 1-5).

A one-way ANOVA was conducted for test trial totals, and a separate ANOVA

was conducted for test trial averages. Effects for trial totals and averages were not

significant, however the averages for trial totals in the saline (M = 26.42) and nicotine (M

= 21.31) conditions indicate a clear trend toward significance (see Figure 5 for a graph of

Phase 1 test trial averages). Additionally, as in the single-phase cohort, five one-way

ANOVAs were conducted for each test trial in Phase 1. Effects of condition for trials 1


through 5 were not significant, indicating that the nicotine did not produce a significant

difference in latencies in each trial.

Phase 2

Statistical analyses for Phase 2 acquisition were conducted in the same fashion as

in Phase 1 and the single-phase cohort. A repeated measures analysis using a 2 (group) x

5 (days) mixed design ANOVA was conducted with trial totals across the 5 days of

acquisition. Trial totals were calculated by adding the latencies of trials 1 through 5 for

each rat in each day. The independent variable was condition and the dependent

variables were the trial latencies.

In the analysis of trial totals, an effect of condition across days was not seen,

indicating that nicotine did not have a significant effect on trial totals across acquisition

days 1 through 5. A significant effect of training was seen for trial totals across days,

F(4, 116) = 9.86, p < .001. This demonstrates an effect of the task, as latencies in both

conditions decreased as a result of training.

The performance between groups on each acquisition day was assessed using a 2

(group) x 5 (trials) mixed design ANOVA, which was conducted with repeated measures

across the 5 trials per day. An effect of condition (nicotine or saline) for individual days

1 through 5 was not seen, indicating that in the second phase of acquisition, condition did

not have a significant effect on trial latencies. The effects of training for day 1, F(4, 116)

= 8.86, p < .001, and day 2, F(4, 116) = 4.82, p < .001, were significant, indicating a

decrease in latencies due to learning of the task (see Figure 6 for a graph of Phase 2

acquisition trials as compared to Phase 1 acquisition trials). The effects of training for


days 3, 4, and 5 were not significant, indicating again that the bulk of learning occurred

in the early days of acquisition.

A paired samples t-test was conducted to determine differences between the first

trial in Phase 1 acquisition and the first trial in Phase 2 acquisition. This provided insight

as to whether previous training had a significant effect on trial latencies in Phase 2

acquisition, regardless of group. The difference between the two trials was significant,

t(1, 30) = 9.39, p < .001, indicating that trial latencies were significantly decreased in

Phase 2 acquisition when compared to Phase 1 (this result is also demonstrated in the

graphs in Figure 6). For Phase 2 acquisition, a one-way ANOVA was also conducted to

view possible differences in trial latencies across the four drug treatment groups, N/N,

N/S, S/N, or S/S. The independent variable was the drug treatment group and the

dependent variables were the trial latencies. Effects of group for days 1 through 5 were

not significant, indicating no significant differences between groups in the Phase 2

acquisition trials.

Similar to the test trial analyses conducted in the single-phase cohort and Phase 1,

a one-way ANOVA was conducted for test trial totals, and a separate ANOVA was

conducted for test trial averages. The independent variable was condition (rats that

received either nicotine or saline in Phase 2), and the dependent variables were trial

latencies. The effect of condition on trial totals was significant, F(1, 29) = 9.16, p = .005,

demonstrating that the nicotine treated adult rats had lower overall trial latencies

compared to saline treated rats. A significant effect of condition was also seen in trial

averages, indicating lower trial latency averages in nicotine rats than in saline rats, F(1,

29) = 9.16, p = .005 (see Figure 7 for a graph of Phase 2 test trial averages). This shows


that the nicotine rats demonstrated increased learning of the task when compared to saline

rats.

Additionally, five one-way ANOVAs were conducted for each test trial in Phase

2, with condition (nicotine or saline) as the independent variable and trial latencies as the

dependent variable. Significant effects of condition were found for trial 1, which had the

same start location as the acquisition trials, F(1, 29) = 6.19, p = .019, trial 4, F(1, 29) =

6.59, p = .016, and for trial 2, F(1, 29) = 4.12, p = .05. This indicates that in these testing

trials, the nicotine had a positive effect on the cognition of the older rats. The effects in

trials 3 and 5 were not significant. These results, coupled with the results from the test

trial totals and test trial averages, demonstrate that nicotine administration led to lower

trial latencies in adult rats.

A one-way ANOVA and LSD follow-up tests were then conducted to view

differences between the four drug treatment groups, N/N, N/S, S/N, and S/S, in the Phase

2 test trials. The independent variables were the four groups with varying drug

conditions, and the dependent variables were the trial latencies. Trial totals for each

group were calculated, and these scores were used in the ANOVA. A significant effect

of group was found for trial totals, F(3, 27) = 3.03, p = .047. Least Square Difference

(LSD) post-hoc tests conducted at a significance level of p < .05 confirmed that groups

receiving nicotine responded more quickly in the MWM due to decreased trial latencies.

LSD tests indicated that for trial totals, the N/N group was significantly faster than the

N/S group (mean difference = 8.57) and the S/N group was significantly faster than the

N/S group (mean difference = 9.31).

Five one-way ANOVAs were conducted using the four groups as the independent


variables and the test trial latencies as the dependent variables, in order to view

differences between groups in individual trials. The effects of the groups in individual

trials 1 through 5 were not significant. However, LSD tests indicated a significant

difference between the N/N group and S/S group in Trial 1 (mean difference = .81) and

Trial 4 (mean difference = 2.08), as shown in Figure 8. Though the effects of these were

not significant in the ANOVA, the significant post-hoc test provides more support for the

result that nicotine rats in Phase 2 were faster than saline rats. For a graph demonstrating

the differences between the N/N, N/S, S/N, and S/S test trial averages, see Figure 9.

A one-way ANOVA was conducted for Phase Differences, which was the

difference in latencies for the four groups (N/N, N/S, S/N, S/S) between Phase 1 and

Phase 2. A near-significant effect of condition was found, F(3, 27) = 2.93, p = .051, and

no significant effect was seen for age alone. This indicates an effect of the nicotine as the

rats aged, however the lack of a significant result for age alone suggests that due to the

previous training, there was no significant difference between test trial latencies in Phase

2 versus Phase 1. LSD post-hoc tests indicated a significant difference between the S/N

and N/S groups, showing that the rats receiving saline in Phase 1 and nicotine in Phase 2

(S/N) had significantly lower trial latencies than rats receiving nicotine in Phase 1 and

saline in Phase 2 (N/S) (mean difference = 16.36).

c-Fos Analysis

Separate one-way ANOVAs were conducted for Phase 2 of the dual-phase cohort

and the single-phase cohort to analyze c-Fos expression in the dentate gyrus of the

hippocampus. Here, the independent variables were the drug conditions and brain

sections, while the dependent variable was c-Fos content.


For Phase 2, three one-way ANOVAs were conducted across condition (nicotine

or saline) on c-Fos expression separately for each section of the hippocampus. The three

sections of interest were the rostral, middle, and caudal portions of the dentate gyrus. A

significant effect of condition was seen in the caudal section, F(1, 26) = 10.43, p = .003.

Means indicate that the rats receiving saline in Phase 2 (M = 14.47) had higher c-Fos

content in this section than did the rats receiving nicotine in Phase 2 (M = 9.23). Effects

for the rostral and middle sections were not significant, indicating the largest c-Fos

difference occurred in the caudal hippocampus section. See Figures 10, 11, and 12 for

examples of rostral, middle, and caudal sections of the dentate gyrus for both saline and

nicotine rats. A one-way ANOVA conducted for the single-phase cohort revealed no

significant effect of condition across nicotine and saline rats in any brain section,

indicating that the nicotine did not have an effect on c-Fos content in the adolescent rats.

Three one-way ANOVAs and LSD post hoc tests at significance level p < .05

were also conducted in Phase 2 to view differences between the N/N, N/S, S/N, and S/S

groups in the three hippocampus sections. Here, the independent variables were the four

groups, and the dependent variable was the c-Fos content. Again, the effect of group for

the caudal section was significant, F(3, 24) = 4.14, p = .017, indicating significantly

higher c-Fos expression in the saline rats. LSD post-hoc tests indicated that S/S rats had

significantly more c-Fos than N/N rats (mean difference = 6.08), and than S/N rats (mean

difference = 7.00). No significant effects of group were seen for the rostral and middle

sections.

To view differences relating to age between the Phase 2 rats and the single-phase

cohort, three 2 (age) x 2 (condition) ANOVAs were conducted for the three hippocampus


sections of interest. Phase 2 rats were considered adults and single-phase cohort rats

were adolescents, and they compared by condition (nicotine or saline). Again, a

significant effect of condition was seen in the caudal section of the hippocampus, F(1,

37) = 8.22, p = .007, indicating the saline rats showed more c-Fos expression than the

nicotine rats. Significant effects for age were seen in the rostral section, F(1, 37) = 5.34,

p = .027, and the caudal section, F(1, 37) = 5.78, p = .021, indicating that adolescent rats

showed less c-Fos expression than adult rats. The effect of age in the middle section

indicated a trend toward significance, F(1, 37) = 3.52, p = .069.

BrdU Analysis

No BrdU expression was demonstrated in the dentate gyrus of the hippocampus in

the rostral, middle, and caudal sections examined for the single-phase and dual-phase

cohorts.

Discussion

The hypothesis that rats receiving nicotine would be significantly improved in the

test trials of the Morris water maze task when compared to rats receiving saline was

partially supported: The adult rats demonstrated a clear improvement in the task after

nicotine exposure, while the adolescent rats did not demonstrate such improvement. As

expected, in Phase 2 testing the N/N rats were significantly improved at the task when

compared to the three other groups, and the S/N rats were improved compared to the S/S

and N/S groups that received saline in Phase 2. The hypothesis that previous training in

the Morris water maze would lead to lower trial latencies overall in Phase 2 of acquisition

was supported, as all rats (regardless of drug condition) demonstrated much faster

reaction times than they did in Phase 1. However, the hypothesis that nicotine rats in


Phase 2 of acquisition would re-acquire the task faster than the saline rats was not

supported, indicating that the nicotine did not have an effect on the re-acquisition of the

task from the same start location.

The hypothesis regarding c-Fos in the dentate gyrus of the hippocampus was not

supported, as we expected to see an increase in c-Fos in the nicotine rats. There was,

however, a significant increase in c-Fos in the dentate gyrus of the rats that received

saline in Phase 2, as the S/S group had a much higher amount of c-Fos than the N/N and

S/N groups. Additionally, it was found that adult rats demonstrated more c-Fos

expression in the hippocampus than adolescents from the single-phase cohort. The

hypothesis that there would be an increase in BrdU in the dentate gyrus of the nicotine

rats was not supported, as we were not able to obtain any BrdU expression in our tissue.

This could have been due to a number of methodological factors, which will be expanded

upon later.

Behavioral Effects of Nicotine

In adolescent rats, contrary to our original hypothesis, there were no significant

differences between the nicotine and saline rats. These rats were approximately two

months old, and still at an early stage of development. It is possible that the lack of a

significant difference in the acquisition and test trials for the adolescent rats (in both the

single-phase cohort and Phase 1 of the dual-phase cohort) is due to the fact that a nicotine

dose of 0.2 mg/kg/day is not enough to demonstrate such an effect in rats of this age. It

has already been demonstrated that adolescent rats metabolize drugs faster than adult rats,

so it is possible that in order to see a change in trial latencies, adolescent rats would need

to be administered a higher dose of nicotine (French et al., 2006). This conclusion


coincides with a previous study that gave adolescent and aged rats a high nicotine dose of

0.5 mg/kg/day, but demonstrated an improvement in the working memory of the

adolescent rats only (Levin & Torry, 1996). This provides evidence for a possible dosing

threshold in both adolescents and adult rats.

In Phase 2 of testing, when the rats in the dual-phase cohort were approximately 6

months old and thus considered adults, complete support of our hypothesis was

demonstrated. The results from the test trials for this phase, in which rats were to find the

hidden platform from varying start locations, demonstrated a clear decrease in trial

latencies in the nicotine rats as compared to the saline rats. This follows from studies that

have demonstrated nicotine as having a pronounced effect on aged rats (French et al.,

2006; Socci et al., 1995). Additionally, previous studies have demonstrated the

importance of allowing a few days for the drug to take effect before starting acquisition

trials (Bernal et al., 1999). Because the rats in this study were given a week prior to

acquisition to acclimate to the drug, it can be concluded that the effects of the nicotine in

the Morris water maze in Phase 2 of testing were due to the nicotine itself and not some

other factor. It can also be concluded that the improvement in the Morris water maze

task among adult rats receiving nicotine was not due to an increase in locomotion as a

result of the nicotine, as the results demonstrate that nicotine did not have an effect on the

activity levels of the rats. This provides more evidence that the nicotine served to

increase the cognition of the rats, and not merely to increase their locomotion.

Our study is unique in that the rats in the dual-phase cohort were given varying

drug schedules that allowed for four separate groups to be examined—this allows us to

draw conclusions regarding the level of impact the nicotine has on the rats’ ability to


learn the Morris water maze task. In Phase 2, our hypothesis was supported because we

found that the N/N rats had significantly lower latencies in the test trials than the N/S rats

and the S/S rats. This provides evidence that learning in the Morris water maze was

state-dependent on the nicotine—when rats received nicotine in Phase 1 and then nicotine

again in Phase 2, they performed very well in the test trials in Phase 2. When rats

received nicotine in Phase 1 and saline in Phase 2, they demonstrated impairment when

compared to all other rats (including the S/S group). This suggests that when rats are

deprived of nicotine and then faced with a task they learned while under the influence of

nicotine, the absence of nicotine leads them to perform worse on the task than they would

have if they had never received nicotine in the first place. In Phase 2 of this study,

however, the rats were faced with the same exact task under identical conditions. It is

possible that if faced with a slightly different task, or the same task in a different

environment, the deficits would not be as pronounced. Thus, the extent to which learning

is state-dependent on nicotine is an area that lends itself to further study.

These results also demonstrate the importance of previous training in conjunction

with the drug treatment, as the re-acquisition of the task in Phase 2 demonstrated lower

latencies overall than did the acquisition in Phase 1. Clearly the previous training helped

the animals learn the task, most likely due to Long Term Potentiation (LTP) in the

hippocampus. Because the training led to an increase in neuronal firing and expression,

the rats were able to perform better during the second phase of acquisition. This follows

from previous research that highlights early training as an indicator of long-term

memory—because the rats remembered the task from the previous acquisition phase, they

were able to re-acquire it much more quickly in Phase 2 (Hansalik et al., 2006).


Because the nicotine did not produce a significant difference in acquisition trials

in Phase 2, it can also be concluded that the previous training in the maze from that start

location is what allowed the rats to recall the task much more quickly than they did in

Phase 1. Additionally, the test trial latencies in Phase 2 did not differ overall from test

trial latencies in Phase 1—thus, the main difference in latencies from one phase to the

next was seen in the acquisition trials. The real difference between nicotine and saline

rats was seen in the Phase 2 test trials, in which the rats had to remember the task relying

only on spatial cues. In this instance, it is clear that the nicotine improved the spatial

learning and memory retention in the rats.

Neuronal Effects of Nicotine

Initially, we hypothesized that there would be an increase in c-Fos expression in

the adult rats in Phase 2 as a result of the nicotine expression. Though this hypothesis

was not supported, a significant result in the opposite direction was found. The saline

rats in Phase 2 demonstrated an increase in c-Fos in the dentate gyrus of the hippocampus

when compared to nicotine rats. Though this did not follow with our hypothesis

regarding c-Fos as an indicator of neuronal change, it follows from previous research

investigating c-Fos as an indicator of stress (Kovacs, 1998). Previous research

demonstrating an increase in c-Fos as a result of nicotine exposure has shown that higher

doses (of up to 2 mg/kg) of nicotine lead to this expression (McCormick & Ibrahim,

2007; Ren & Sagar, 1992). Because the dose used in the present study was quite low (0.2

mg/kg), it is possible that this was not enough nicotine to produce a c-Fos increase in the

nicotine rats. That being said, the increase in c-Fos in saline rats could instead be due to

the increased amount of stress the saline rats were under. Because the results show that


saline rats demonstrated significantly higher latencies in the test trials of the MWM task,

it is possible that being in the maze for a longer period of time led to more stress in these

rats when compared to the nicotine rats that were in the maze for much shorter periods of

time. This follows from studies examining the effects of swim stress on rats—rats forced

to swim (usually for 15 minutes) with no escape platform show high stress levels as

indicated by increased c-Fos expression in the brain (Lino-de-Oliveira et al., 2006).

Though it seems this will often be a confound in an experiment like this, it would

be possible to use a different Morris water maze method that would eliminate the

possibility of increased stress in the rats with higher trial latencies. In this experiment,

the platform was present at all times during the test trials. A future experiment could

employ the use of a camera suspended over the maze during test trials—the platform

would be removed, and the amount of time the rats spend in each quadrant would be

assessed instead. The amount of time spent in the quadrant that contained the platform

during acquisition would be the measure of spatial learning. This way, all rats would be

in the maze for the same amount of time (generally 60 seconds), and increased stress as a

result of higher swimming times would not potentially skew the c-Fos results.

Though we expected to see an increase in cell proliferation in the dentate gyrus of

the hippocampus as evidenced by BrdU, no such staining was obtained. Due to a number

of studies demonstrating an increase in neurogenesis in the hippocampus as evidenced by

BrdU expression after nicotine administration and spatial learning, the lack of BrdU

staining is most likely because of a problem with our procedure (Dalla et al., 2007;

Dobrossy et al., 2003). It is possible that the number of injections was not enough to lead

to significant BrdU staining in the hippocampus. Previous studies employing the use of


BrdU generally use three or more injections of 50 mg/kg BrdU, or they use one or two

injections of an extremely high concentration of BrdU (Dalla et al., 2007; He & Crews,

2007; Scerri et al., 2006). In this study, we only injected the rats twice with a

concentration of 50 mg/kg BrdU, and it is possible that in order for the nicotine or

training to have an impact on BrdU expression we would have needed more injections.

In addition to administering more BrdU injections, it would be beneficial in the

future to modify the injection schedule so the timing coincides with the time when the

most learning takes place. A previous study divided the learning process in the Morris

water maze into an early phase, in which most of the learning occurs, and a late phase, in

which the latencies decrease and level off (Dobrossy et al., 2003). It was found in the

study that the early phase of learning did not produce significant cell proliferation

potentially due to increased stress of a new task, but that the late phase of learning did

demonstrate increased cell proliferation as evidenced by BrdU (Dobrossy et al., 2003).

However, the study employed fewer acquisition trials per day, and they only looked at

acquisition (what they termed “learning”) trials. The timing of our injections was right

before the last day of acquisition and before the first day of testing—it is possible that

because at this point the rats had already significantly learned the task, there were not

enough newly-formed cells to demonstrate BrdU incorporation in the hippocampus. It

can also be argued that the later acquisition trials in our study were less hippocampal-

dependent because the rats had already been exposed to a number of trials from the same

start location. If this is the case, it makes sense that there was no BrdU expression in the

hippocampus after these trials. Instead, administering injections during the first

acquisition days in which the bulk of the learning occurs would most likely lead to an


increase in BrdU expression.

Due to our knowledge of the effects of nicotine at the neural level, it can be

concluded that despite the lack of c-Fos and BrdU expression in the nicotine rats, the

nicotine still had an effect on the nAChRs in the rats’ brains. The hippocampus is known

to be highly involved in memory and learning due to LTP, which is an increase in

synaptic effectiveness. Because the behavioral data demonstrated that the adult rats

treated with nicotine were significantly improved in the Phase 2 test trials, we can reach

the conclusion that the nicotine was binding to nAChRs and stimulating the release of

neurotransmitters in the brain. This led to an increase in neuronal firing, as indicated by

the enhancement in their cognition as related to the Morris water maze task.

The results of this study follow from previous studies investigating the effects

nicotine has on the hippocampus. It has been shown that nicotine facilitates synaptic

activity and can increase LTP in the hippocampus via tasks involving memory and

learning (Rezvani & Levin, 2001). Nicotine’s positive effect on LTP has also been

supported by studies investigating nicotine as a neuroprotective agent in rats that have

been exposed to significant stressors (Aleisa et al., 2006). In the case of Alesia and

colleagues (2006), nicotine was shown to normalize LTP and counteract working

memory impairments that were induced by stress. Nicotine has also been shown to

attenuate working memory deficits as induced by certain drugs, again providing evidence

that nicotine is neuroprotective and plays a significant role in LTP in the hippocampus

(Levin, Bettegowda et al., 1998).

While there is significant evidence that nicotine leads to increased neuronal firing

and LTP in the hippocampus as a result of tasks involving memory and learning, it is


possible that the site of neuronal change could be elsewhere in the brain, or in addition to

these hippocampal effects. Studies have shown that nAChRs are also present in other

brain areas, such as the amygdala, the prefrontal cortex, the thalamus, and the motor

cortex (Levin, McClernon et al., 2006; Mansvelder et al., 2006). It would be beneficial

to investigate these brain areas with respect to nicotine exposure and working memory

tasks, as this would provide a more complete view of the neural mechanisms of nicotine.

Limitations

The limitations of this study mostly involved the newness of the procedure. The

osmotic mini pumps had never been used in this laboratory before, and due to some

complications in the first round of surgeries a few rats lost their pumps. This was most

likely due to poor placement of the pumps, as the first batch had the releasing end of the

pump facing the wound. The excessive scratching that led to the loss of the pumps could

have been due to the nicotine aggravating the wounds before they healed. After the first

round of surgeries all other surgeries were completed without incident, providing further

evidence that the pump placement was the reason for the excessive scratching and

subsequent pump loss.

The use of BrdU was another limitation in this study. It did not immediately go

into solution, so based on the findings of previous studies we heated the BrdU

significantly in order to help it dissolve (Hume & Keat, 1990). We later found, however,

that heating BrdU might actually decrease the likelihood that it will incorporate into new

neurons. Despite this, the previous literature that used the heating technique did still see

BrdU expression—thus, it was most likely a problem with the timing of our injections

that led to the lack of expression (Hume & Keat, 1990). The results from the present


study suggest that the ideal time to inject BrdU is when learning is most evident, which

would have been during days 1 and 2 of the acquisition trials.

Another potential limitation in this study could have been in the use of the Morris

water maze. There are a few different ways the Morris water maze can be used to

demonstrate spatial learning, and the procedure used in this study may be unusual

compared to other, more widely used procedures. Because the rats were to be exposed to

the maze four months after Phase 1 of testing, we wanted to ensure that the rats learned

the task as well as possible during Phase 1. For this reason, we chose to have five days of

acquisition, with five trials from the same start location each day. As the results indicate,

the rats learned the spatial cues by the end of acquisition trials in both Phase 1 and 2. It

can be argued that by the end of the acquisition trials the task was no longer

hippocampal-dependent because the start location remained the same; however the good

performance of the rats in the test trials indicates that the hippocampus was still highly

involved in the task.

There have been previous studies using the Morris water maze as both a

hippocampal-dependent and independent task. It has been found that when the task

involves spatial learning, such as in a traditional Morris water maze study in which rats

must locate a hidden platform, it is dependent upon the hippocampus (Teather, Packard,

Smith, Ellis-Behnke, & Bazan, 2005). This has been evidenced by an increase in c-Fos

in this brain area after such a task (Teather et al., 2005). When a task is due to a

stimulus-response “habit,” such as in a Morris water maze task in which the platform is

visible, the task can be completed independent of the hippocampus, with more reliance

on the dorsal striatum. In the case of habitual learning, the increase in c-Fos is seen in the


dorsal striatum, and not the hippocampus (Teather et al., 2005). Based on the findings of

this study, it is possible that the later acquisition trials in the present study were more

habitual, and that they relied on the dorsal striatum more than the hippocampus. Yet

these findings still provide support for the test trials as being hippocampal-dependent

because the rats had to find the hidden platform from unfamiliar start locations around the

maze.

Many Morris water maze studies also employ the use of a video camera in order

to assess the amount of time rats spend in each quadrant when the platform is removed

(Bernal et al., 1999; Socci et al., 1995). Because this kind of technology was not

available to our laboratory at the time of Phase 1 acquisition and testing, we chose to

keep the platform in the maze for all parts of the experiment. Therefore, it was necessary

to have all acquisition trials begin at the same start location, and to test the learning of the

task using 5 different start locations around the maze. Although this is a more traditional

method of assessing Morris water maze spatial learning, it still yielded significant results

and demonstrated that, after 5 days of acquisition and 1 test day at varying start locations,

rats learned the task as evidenced by a significant decrease in trial latencies (Morris,

1984).

This study may have also been limited by low statistical power. Though it began

with 50 subjects and an even number of nicotine and saline rats in each group,

complications with the surgeries and pumps caused these numbers to decrease, and

ultimately the data from four subjects were lost before the close of the experiment.

Though the results from the test trials were significant, it is possible that any differences

in the adolescent rats in Phase 1 were too subtle to be demonstrated by the small number


of rats used in each group. Additionally, the complicated nature of the study design led

us to perform many statistical analyses, which could have contributed to the possibility of

Type I error. Despite this, the conclusions made were based largely on the results of tests

using trial totals and averages, and the separate ANOVAs conducted were simply to view

potential differences within the days of acquisition, and within the individual test trials.

It may be necessary in the future, however, to alter the experimental design to allow for

fewer statistical analyses to avoid this potential problem.

Implications

The results of this study lead to a number of implications that can be translated to

human studies of nicotine’s cognitive-enhancing properties. The finding most relevant to

human research is that nicotine did enhance the cognition of the adult rats. This

conclusion provides continuing support the use of nicotine as a therapeutic alternative for

people suffering from diseases affecting cognition, including Alzheimer’s disease. It also

paves the way for future studies in this area that will give us even more information about

the cognitive enhancing properties of nicotine, and how it can be best utilized for

treatment of such disorders.

The fact that we tested adult rats that were 6 months old as opposed to “aged” rats

that were up to 24 months old could be a potential limitation of this study because it

makes it difficult to compare to previous literature. Most studies examining the

differences between young and aged rats use aged rats because it is easier to relate to

human studies of elderly people, particularly ones with age-associated memory

impairments. The fact that we used adult rats, however, is also beneficial because of the

lack of research done on rats of this age group—we were able to demonstrate that adult


rats benefited from a small amount of nicotine, which provides important results

regarding a potential dosing threshold between adolescent and adult rats. Previous

studies have only been able to demonstrate that adolescent rats need a higher dose than

aged rats—the current study is able to narrow that age gap, and it suggests that there is a

point between the age of 2 months and 6 months where the dose of nicotine can be

decreased dramatically.

This translates to human studies involving nicotine by suggesting that if adults are

facing a disease affecting their cognition, they do not need a dose as high as an

adolescent might need in order to delay the onset of this disease. This could dramatically

decrease the potential dangers of employing the use of nicotine patches in adult patients,

because they would not be exposed to extremely high doses of nicotine that could lead to

adverse side effects or increased likelihood of addiction. Instead, future studies of

nicotine’s use in adults facing cognitive impairments could begin using a relatively low

dose of nicotine—if that does not produce the desired effects, then a higher dose might be

tested. But the fact that the adult rats in this study benefited from the same low dose that

has been shown to improve cognition in aged rats provides evidence that adult humans

may also benefit from the same dose as the elderly (Socci et al., 1995). Human studies of

nicotine employing the use of nicotine patches delivering 0.9 mg/kg per hour (considered

a moderate dose) to patients in their late 70’s have demonstrated a clear improvement on

tasks of cognition (Wilson et al., 1995). Based on the results of the current study, it

would most likely not be necessary to give an adult a higher dose than that in order to

improve cognition.

The results regarding the dose in this study are also important in demonstrating


that cigarette smoking is not a safe therapy to use for people with diseases affecting

cognition. Though it has been hypothesized that many patients with schizophrenia and

ADHD self-medicate with cigarettes to obtain nicotine, the results of this study imply that

this practice can actually have adverse effects upon the patients due to the very high dose

of nicotine and the other harmful ingredients in cigarettes (Levin, McClernon et al., 2006;

Winger et al., 2004). Cigarette smoking can actually impair cognition because the non-

nicotine ingredients can restrict blood flow to the brain and lead to cell death. Also, the

very high amount of nicotine in cigarettes can actually decrease cognition due to receptor

desensitization (Winger et al., 2004). Thus, nicotine should really be administered by

itself and in a low dose to decrease the potential negative side effects of the drug.

It can also be concluded from the current study that the performance of the adult

rats in the Morris water maze was state-dependent on the nicotine. This means that after

being exposed to nicotine in conjunction with the spatial learning task, it was necessary

to provide nicotine to the rats in any future exposures to the same task. This is why the

group that received nicotine in Phase 1 was impaired in Phase 2 when they received

saline—their learning of the task was dependent on the nicotine, and thus their trial

latencies without it were much higher than rats in all other groups. It is possible,

however, that the detriment in the N/S group might not have been evident if they were

exposed to a different task, or the same task under new circumstances. In the current

study, it is not possible to separate the state-dependent effect from the potential long-term

benefits because we used the same spatial learning task to assess memory retention in

both phases.

Despite the current data regarding the state-dependent effects of nicotine, it is not


likely that nicotine cessation would produce such profound deficits in humans after

learning a task. In a study examining the potential cognitive deficits due to withdrawal

from the nicotine in cigarettes, impairments on tasks of cognition were seen a few hours

following cessation of the drug (Hatsukami et al., 1989). However, it was mentioned that

other studies using similar methods demonstrated that test scores on tasks of cognition

after quitting were higher than scores obtained when the same participants were engaged

in cigarette smoking (Hatsukami et al., 1989). This suggests that the decline in the study

done by Hatsukami and colleagues (1989) may not have been wholly due to the nicotine

cessation, but also due in part to lasting negative effects of the combination of ingredients

in cigarettes. Additionally, the study did not test their participants beyond 24 hours after

quitting—this would be crucial for conclusions that nicotine cessation produces long-

term cognitive deficits. Because this was not done, it is not possible to make this kind of

assertion.

In order to further examine whether cognitive tasks are state-dependent on

nicotine in humans, it would be necessary to perform follow-ups to the studies on

nicotine’s effect on human cognition. There have been a number of studies

demonstrating that a chronic (or even acute) dose of nicotine improves memory and

learning in Alzheimer’s patients, however it would be beneficial to re-examine these

patients after a significant period of time with the same tasks to see if the absence of

nicotine has a negative effect on their ability to perform the task (Jones et al., 1992;

Sahakian et al., 1989; Wilson et al., 1995). This would provide more concrete evidence

for the state-dependent effects of nicotine in humans.

The conclusion from the current study that c-Fos was increased in the


hippocampus potentially due to the elevated stress levels of the saline rats has interesting

implications for human studies. It suggests that humans who are facing the beginning

stages of cognitive decline could be under significantly more stress than people not

undergoing the same decline. Though more research would need to be done in this area,

it can be hypothesized that if humans are given nicotine to enhance their cognition at

these beginning stages, they may be under much less stress because they would not have

the same level of difficulty performing everyday tasks. Additionally, this provides some

evidence that perhaps nicotine has anxiolytic effects. This is a result that would benefit

from further study, because it provides more support for the development of a therapeutic

treatment for such diseases that enhances cognition and delays the onset of marked

cognitive decline.

Future Studies

Because this is a preliminary study, it lends itself to replication on a larger scale.

Increasing the number of subjects in each group would be extremely beneficial, as the

increase in power would potentially demonstrate subtle changes between groups,

particularly in the adolescent rats. There were many trends toward significance, and it is

assumed that with more subjects, there would be more clear indications of significance.

Replicating this study would also be beneficial as many of the problems encountered

would be anticipated and resolved before they occurred. It is now known how to

properly perform the surgeries, so this would not be a complication, and the

administration of BrdU would be handled differently. More injections of BrdU would be

administered, and the timing of these injections would be altered so that the rats would be

injected during the first two acquisition days. These days were when the bulk of the


learning occurred, so it is most likely that injections during these two days would lead to

an increase in BrdU expression in the hippocampus.

This study also paves the way for further research in this area. We demonstrated

that a nicotine dose of 0.2 mg/kg showed a significant decrease in the trial latencies of

adult rats in the Morris water maze, but this dose did not demonstrate a decrease in

latencies in adolescents. In the future, this experiment could be replicated by giving a

group of adolescent and adult rats a higher dose of nicotine (for example, 0.4 mg/kg), in

order to determine whether this dose improves the performance of the adolescents, and

whether it impairs the performance of the adults.

Because the results regarding c-Fos expression in the dentate gyrus of the

hippocampus were unexpected, further research regarding nicotine’s effect on c-Fos

expression in the rat brain would be beneficial to our understanding of this relationship.

In order to demonstrate whether the increased c-Fos expression was due to the stress of

having higher trial latencies, it would first be useful to look at c-Fos as a result of Morris

water maze training alone—then the results could be separated into rats with longer

latencies and rats with shorter latencies, to see if the rats that spent more time in the maze

had more c-Fos expression. It would also be beneficial to examine c-Fos expression in

the rat hippocampus as a result of nicotine when stress is not a factor. This would simply

involve administering nicotine for a certain length of time and then examining c-Fos

expression in the hippocampus after that time. If there is c-Fos present in the nicotine

rats, it can be concluded that nicotine increases c-Fos expression when stress is not a

factor.

Because the present study examined the hippocampus only, it would certainly be


beneficial to examine other brain areas that are linked both with memory and learning,

and also with stress. This would be useful to provide conclusions regarding the effects of

the nicotine at the neural level. Because nicotine can lead to increased attention in

humans, it has been proposed that nicotine may also have an effect on the prefrontal,

parietal, and occipital cortex, as well as the amygdala, and the hippocampus (Mansvelder

et al., 2006). Additionally, the thalamus and the motor cortex have also been shown to

demonstrate a high number of nAChRs, which could also have implications regarding

nicotine’s effect on the human as well as the rat brain. Replicating this study and

examining c-Fos or BrdU expression in some of these brain areas could be extremely

beneficial for this field of research, as the largest amount of work has been done with the

hippocampus. Paying closer attention to the brain areas about which less is known

regarding nicotine’s effects could lead to some important conclusions that could be

carried over to human studies of nicotine. Examining nicotine’s effect on the motor

cortex could be particularly interesting when testing rats in motor tasks like the Morris

water maze, because if nicotine stimulates neuronal growth in this brain area it could

have a significant impact on the ease with which the rats acquire the task.

Conclusions

The current study provides support for the use of nicotine as a therapeutic agent in

diseases affecting cognition. Because of nicotine’s direct stimulation of nAChRs in the

human brain, it would be a more effective alternative to the current treatments available

for diseases like Alzheimer’s, which provide minimal symptom relief. Though a

considerable amount of information is known about nicotine’s effects on the

hippocampus, it is important that future studies examine nicotine’s effects on different


brain areas in order to provide a more comprehensive view of its neural mechanisms.

Once this knowledge is attained, it will hopefully lead the use of nicotine as a therapeutic

drug—this has the potential to dramatically increase the quality of life for people

suffering from diseases affecting their cognition.


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Table 1 _______________________________________________________________________ Drug Treatment Groups _______________________________________________________________________ Group Phase 1 Phase 2 Number of Subjects N/N Nicotine Nicotine 7 N/S Nicotine Saline 7 S/N Saline Nicotine 9 S/S Saline Saline 8 _______________________________________________________________________


Table 2

_______________________________________________________________________

Experiment Schedule

_______________________________________________________________________

Experimental Day 1 2 3 4 5 6 7 8

Phase 1 A A A A A - - T

Phase 2 (and single-phase A A A A - A T cohort) _______________________________________________________________________

Note. A = Acquisition day, T = Test day, dash (-) = no trials


Figure Caption

Figure 1. Morris water maze start locations and platform placement. Figure 2. Mean trial latencies for single-phase cohort acquisition.

Figure 3. Mean trial latencies for single-phase cohort test trials.

Figure 4. Mean trial latencies for Phase 1 acquisition.

Figure 5. Mean trial latencies for Phase 1 test trials.

Figure 6. Mean trial latencies for Phase 2 acquisition compared to mean trial latencies

for Phase 1 acquisition.

Figure 7. Mean trial latencies for nicotine and saline rats in Phase 2 test trials.

Figure 8. Mean trial latencies for N/N and S/S rats in Phase 2 test trials.

Figure 9. Mean trial latencies for N/N, N/S, S/N, and S/S in Phase 2 test trials.

Figure 10. Photograph of c-Fos expression in saline and nicotine rats in the rostral

section of the dentate gyrus (arrows point to examples of c-Fos particles).

Figure 11. Photograph of c-Fos expression in saline and nicotine rats in the middle

section of the dentate gyrus.

Figure 12. Photograph of c-Fos expression in saline and nicotine rats in the caudal

section of the dentate gyrus.


Figure 1.


Figure 2.

05

1015202530354045

T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5Day 1 Day 2 Day 3 Day 4 Day 5

Acquisition Trials

Tim

e (s

)

NicotineSaline


Figure 3.

0

2

4

6

8

10

12

1 2 3 4 5

Start Location

Tim

e (s

)

NicotineSaline


Figure 4.

0

5

10

15

20

25

30

35

40

T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5Day 1 Day 2 Day 3 Day 4 Day 5

Acquisition Days

Tim

e (s

)

NicotineSaline


Figure 5.

0

1

2

3

4

5

6

7

1 2 3 4 5

Start Location

Tim

e (s

)

NicotineSaline


Figure 6.

Phase 1 Acquisition Trials

Phase 2 Acquisition Trials

0

5

10

15

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30

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T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5

Day 1 Day 2 Day 3 Day 4 Day 5

Acquisition Trials

Tim

e (s

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NicotineSaline

0

5

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T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5

Day 1 Day 2 Day 3 Day 4 Day 5

Acquisition Trials

Tim

e (s

)

NicotineSaline


Figure 7.

0

1

2

3

4

5

6

7

8

1 2 3 4 5

Start Location

Tim

e (s

)

NicotineSaline


Figure 8.

0

1

2

3

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5

6

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1 2 3 4 5

Start Location

Tim

e (s

)

N/NS/S


Figure 9.

0

1

2

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N/N N/S S/N S/S

Drug Treatment

Ave

rage

Tim

e (s

)


Figure 10.

Saline

Nicotine


Figure 11.

Saline

Nicotine


Figure 12.

Saline

Nicotine

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